Molecular Beacons in Lipoplex Nanoparticles for Extracellular Vesicles Based Cancer Diagnosis

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Molecular & Cellular Analysis Technologies
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AbstractEarly detection is one of the keys to winning the war on cancer. Because current imaging and tissue biopsybased diagnosis are invasive, expensive, and in some cases dangerous, they are impractical as an early cancerscreen, as well as therapeutic and recurrence monitoring. Liquid biopsy is an exciting new form of biopsy thatcan complement cancer detection assays from the analysis of bodily fluids such as blood or urine because it isnon-invasive, easier, safer, and more cost effective. With regards to body fluids based cancer detection andmonitoring, blood analysis is the most reliable source, with three types of analysis emerging as possibleapproaches for detection- circulating tumor cells (CTCs), cell free DNA (cfDNA), and extracellular vesicles (EVs).CTCs have been investigated by many researchers and companies in the past 30 years. Many technologies areavailable for their capture and identification. However, CTCs are rare, particularly in early stage cancer patients(<5 CTCs in 1 mL blood), making quantitative analysis difficult and less reliable. Many cancer patients also donot show CTCs in blood because of endothelial-to-mesenchymal-transition in circulation. Today, CTC basedcancer diagnosis has not been widely used in clinic. Cell free DNA (cfDNA) has gained its success in noninvasiveparental testing (NIPT). But the content of DNA targets from dead target cells such as early stage cancer is low,which requires PCR for amplification and Sequencing for identification. Consequently, the procedure is costly(>$1,000 per test in NIPT). cfDNA for early stage cancer detection is not proven, and many cancers are notinitiated by DNA mutations. Extracellular vesicles (EVs) are abundant in blood (1012 ? 1014 in 1 mL blood), makingquantitative analysis easier. Coding (e.g. mRNA) and non-coding (e.g. microRNA and lncRNA) RNAsencapsulated in EVs and membrane proteins on the surface of EVs can serve as good disease biomarkers.They also play important roles in disease metastasis and relapse, making the capture and characterization ofindividual EVs, not average information from all EVs in the sample as in the existing methods, highly valuable indisease diagnosis and medicine development.Many cancer types including pancreatic cancer have very high mortality rates because it is difficult to detect theirpresence in the early stage. Current biomarkers for pancreatic cancer such as serum CA-19-9 protein face highfalse positive and negative rates. The proposed studies seek to develop a robust and user-friendly novelmolecular beacon in lipoplex nanoparticles for extracellular vesicles based cancer diagnosis and validateits clinic potential by detecting Glypican 1 mRNA and KRAS mutation in extracellular vesicles frompancreatic cancer patient serum samples for imminent use in the fields of early cancer diagnosis andrelapse monitoring. The specific Phase I aims and milestones of this project are: Specific Aim 1: Design andtesting of a toehold initiated (Ti) and catalyzed hairpin DNA circuit (CHDC) CLN-MB which are highlystable, specific and sensitive for GPC1 and KRAS mutation genes. Milestones: (i) Detection of GPC1 andKRAS mutation mRNAs in EVs secreted from <2x104 pancreatic cancer cells in 1 mL blood with >95%repeatability; (ii) lyophilized CLN-MB nanoparticles for >6 months shelf-life and <10% performance variation; (iii)<10% variation in100 assays using a conventional microscope as an analyzer. Specific Aim 2: Dual-sitevalidation of EV GPC1/KRAS mRNA biomarker for early PDAC diagnosis. Milestones: (i) User-friendlyassay with <4 hours assay time; (ii) <10% false positive/negative prediction from >200 pancreatic cancer patientsamples at OSU and MSKCC.